Olanzapine, but not haloperidol, exerts pronounced acute metabolic effects in the methylazoxymethanol rat model

Abstract Aim Widely used second‐generation antipsychotics are associated with adverse metabolic effects, contributing to increased cardiovascular mortality. To develop strategies to prevent or treat adverse metabolic effects, preclinical models have a clear role in uncovering underlying molecular mechanisms. However, with few exceptions, preclinical studies have been performed in healthy animals, neglecting the contribution of dysmetabolic features inherent to psychotic disorders. Methods In this study, methylazoxymethanol acetate (MAM) was prenatally administered to pregnant Sprague–Dawley rats at gestational day 17 to induce a well‐validated neurodevelopmental model of schizophrenia mimicking its assumed pathogenesis with persistent phenotype. Against this background, the dysmetabolic effects of acute treatment with olanzapine and haloperidol were examined in female rats. Results Prenatally MAM‐exposed animals exhibited several metabolic features, including lipid disturbances. Half of the MAM rats exposed to olanzapine had pronounced serum lipid profile alteration compared to non‐MAM controls, interpreted as a reflection of a delicate MAM‐induced metabolic balance disrupted by olanzapine. In accordance with the drugs' clinical metabolic profiles, olanzapine‐associated dysmetabolic effects were more pronounced than haloperidol‐associated dysmetabolic effects in non‐MAM rats and rats exposed to MAM. Conclusion Our results demonstrate metabolic vulnerability in female prenatally MAM‐exposed rats, indicating that findings from healthy animals likely provide an underestimated impression of metabolic dysfunction associated with antipsychotics. In the context of metabolic disturbances, neurodevelopmental models possess a relevant background, and the search for adequate animal models should receive more attention within the field of experimental psychopharmacology.


| INTRODUC TI ON
During the past decades since the introduction of secondgeneration antipsychotic agents, their potential for inducing adverse metabolic effects -diabetes, weight gain, and lipid disturbances -has become well-known. 1 Still, metabolic and cardiovascular adverse events remain a clinical challenge, with high prevalence and non-satisfactory effects of lifestyle and pharmacological intervention alone or in combination. 2,3Despite the extensive research interest, the understanding of the pharmacological and molecular mechanisms mediating the dysmetabolic effects of antipsychotics is insufficient and incomplete, 4,5 also due to the several confounding factors which are limiting the clinical studies, such as comorbidities and concomitant pharmacological treatment.In this context, preclinical models are irreplaceable research tools, allowing for controlled studies and direct examination of networks and tissues. 6This is an essential prerequisite for progress, as clinical studies in the vulnerable patient groups in question should rely on sound hypotheses.[20][21][22] In this regard, a major cause is that modeling of severe psychiatric disorders in animals is inherently challenging.A range of models relying on CNS lesions, pharmacological treatment, genetic deletions, or prenatal exposure have been introduced. 6,23These models differ in theoretical background and the degree to which they meet validity criteria. 24e neurodevelopmental methylazoxymethanol acetate (MAM) model is based on prenatal administration of MAM (gestational day 17), which produces a phenotype characterized by persistent, functional, and neuropathological deficits mimicking schizophrenia in adult offspring. 21,25,26Therefore, the MAM model is an invaluable tool that reproduces the human condition in terms of construct, face, and predictive validity to investigate, in the present study, the significance of prenatal MAM exposure for the response to acute effects of treatment with the atypical antipsychotic olanzapine (OLA) and the typical antipsychotic haloperidol (HAL).These antipsychotics seem to affect metabolic status differentially.HAL has been shown to affect glucose, cholesterol, and TAG in humans, but it is a much less metabolically potent antipsychotic than OLA, an antipsychotic with a very high propensity to adverse metabolic effects. 1,4,27inically observed early weight gain is highly relevant and predicts long-term weight gain during psychotropic treatment. 28,29wever, undesired metabolic features associated with antipsychotics can precede weight gain. 41][32] Adipose tissue dysfunction is a core feature of the pathophysiology of obesity and insulin resistance.At the same time, the adiponectin/leptin ratio is an emerging adipose tissue and metabolic function biomarker, which may serve as a predictor of cardiovascular risk. 33,34Furthermore, in patients suffering from schizophrenia, the adiponectin/leptin ratio represents a potential marker of metabolic syndrome. 35[38][39] Although there is evidence of hormonal dysregulation, its role remains unclear. 40,41e rapid onset of feedback mechanisms may mask dysmetabolic alterations induced by antipsychotics during long-term exposure. 9,42 the present study, an acute setting allowed for the investigation of the direct effects of antipsychotics, independent of weight gain, an approach enabling mechanistic insight and suggesting potential therapeutic strategies.Thus, this study is focused on acute metabolic effects, including peripheral metabolic markers such as adipokine alterations and differences between the two drugs with specific dysmetabolic potential at 3 different consecutive timepoints over 24 h.

| MATERIAL S AND ME THODS
Animal studies are reported in compliance with the ARRIVE guidelines. 43The study aimed to reveal metabolic disturbances induced by systemic OLA or HAL treatment in a well-validated animal model of schizophrenia.Therefore, the use of experimental animals could not be avoided.Female Sprague-Dawley rats were used for consistency with previous literature.This strain and sex are optimal for In the context of metabolic disturbances, neurodevelopmental models possess a relevant background, and the search for adequate animal models should receive more attention within the field of experimental psychopharmacology.

K E Y W O R D S
adipokine, antipsychotic, lipid profile, methylazoxymethanol, schizophrenia | 3 of 13 developing the OLA-induced phenotype, such as increased body weight and food intake. 8,9,36
Environmental conditions during the whole study were constant: relative humidity 50-60%, temperature 23

| Drugs and treatments
Methylazoxymethanol acetate (MAM; Midwest Research Institute, Kansas City, USA) was dissolved in saline and administered intraperitoneally at a 22 mg/kg dose in 1 mL/kg volume on GD 17. Saline was injected to the control group as a vehicle (CTR).The timeline and design of the study are depicted in Figure 1.Both antipsychotics were acutely administered as intramuscular injections designed for human use at 5 mg/kg in 1 mL, and saline was used as vehicle control (VEH): olanzapine (OLA): Zyprexa® (Eli Lilly Nederland BV, Utrecht, The Netherlands) and haloperidol (HAL): Haloperidol® (Gedeon Richter Plc., Budapest, Hungary).The doses of both drugs were chosen based on calculations and extrapolations, considering pharmacokinetic parameters, especially the much shorter half-life of antipsychotics in rodents compared with that in humans.Doses with regard to the route of administration of these antipsychotics were selected based on plasma concentrations and in vivo D 2 -receptor occupancies in rats 8,52 and clinical dosing schemes.

| Sample collection
Approximately 1 mL of blood was collected under short isoflurane (AERRANE®, Baxter S.A., Belgium) anesthesia by retro-orbital puncture one and 4 h after drug administration. 53The intraperitoneal injection of saline supplied the same amount of liquid.Twenty-four hours after drug administration, all rats were sacrificed by decapitation under isoflurane anesthesia to collect blood (for serum).
Dissection was performed after decapitation by wide laparotomy, with sampling from the liver and white abdominal tissues.The samples were frozen at −70°C until laboratory analysis.
The quantification limits of individual analyses were as follows: leptin -100 pg/mL; adiponectin -200 pg/mL; FGF-21 -40 pg/mL; and PAI-1 -298 pg/mL.The quantification limit was used in the case of analysis results under the quantification limit.This was the case of 14 values in leptin analysis, 20 values in FGF-21 analysis, and 12 values in PAI-1 analysis (no adiponectin values were under the limit).The leptin/adiponectin ratio was calculated as previously suggested. 35l analyses were performed using serum samples withdrawn 1 and 4 h after drug administration and in serum samples obtained during dissection 24 h after drug dosing (three timepoints).
Lipid parameters were determined in serum samples obtained 24 h after drug dosing by enzymatic, colorimetric (total cholesterol, HDL), or photometric (LDL, TAG) method (Roche/Hitachi COBAS C®) by Lab Med s.r.o., Brno, Czech Republic.Furthermore, the atherogenic index (AI) was calculated as a ratio of total cholesterol/HDL, and the atherogenic index of plasma (AIP) was calculated as the log(TAG/HDL).The reason for analyzing only the last timepoint was the small amount of blood (and subsequently serum), which was possible to collect in the early timepoints together with a relatively high demand of sample volume for this analysis (approx.50 μL per assayed analyte).

F I G U R E 1
Schematic diagram of the study design.The timeline indicates the design of the study, blood sampling, and all analyses performed at specific timepoints.

| Western blot analysis of white adipose tissue
Sample preparation and Western blot analysis were performed by a standard procedure as presented previously. 54Briefly, approximately 5 mm 3 of adipose tissue was minced using sterile forceps and a scalpel.Fragments were collected to a sterile Eppendorf tube and lysed in 300 μL of SDS lysis buffer (1% SDS, 10% glycerol, 100 mM Tris-Cl pH 7.4).Samples were vortexed vigorously for 1 min, followed by centrifugation at 14,000 rpm for 1 min.This step was repeated three times.
According to the manufacturer's instructions, total protein concentration was determined using DC protein assay (Bio-Rad, USA), and all samples were equalized for protein content.Following the addition of 2-mercaptoethanol (1%) and bromophenol blue (0.01%), the samples were boiled for 5 min at 95°C.An equal amount of protein (30 μg)   was separated by SDS-PAGE.Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, USA).Western blot analysis was performed using standard protocols with the following primary
The prepared specimens were stained using Ventana BenchMark® automated staining (BenchMark ULTRA -Ventana Medical Systems, Roche, Switzerland) according to the manufacturer's instructions.
A blinded observer evaluated all immunostained slides using a light microscope at 400x magnification.The intensity of staining and the percentage of positive cells were assessed at least in five high-power fields of the tissue section.In the majority of samples, the whole area was assessed.The expression of IHC markers was classified into four levels: no expression (0); light expression, less than 25% of positive cells (1); medium expression, 25%-50% of positive cells (2); and very significant expression, more than 50% of positive cells (3).

| Statistical data analysis
Primary data were summarized using the arithmetic mean and standard deviation (±SD).All analyses were calculated using IBM SPSS

| Body weight
All rats were weighed at the time of drug administration.The average BW in the CTR rats was 227 g (±SD 14.47) and in the MAM rats was 192 g (±SD 12.82).The 2 W ANOVA indicated a significant effect of the MAM model (F 1,46 = 79.889,p < 0.001), with significantly lower BW in the MAM rats.No effect of the assignment to a treatment group was observed, and the data had equal variability (data not shown).

| Adipokine levels 1, 4, and 24 h after drug administration
As apparent from Figure 2, with p values presented in the righthand panel, in the serum ELISA assessment of adipokines, the mixed model showed a significant overall effect of the MAM model on leptin (lower than in CTR rats), adiponectin (higher than in CTR rats), and their ratio (lower than in CTR rats).There were numerous significant effects of antipsychotic treatment.Particularly, OLA decreased leptin and leptin/adiponectin ratio and increased adiponectin and FGF-21.HAL increased adiponectin and decreased the leptin/adiponectin ratio.In some cases, these effects were different in the MAM and CTR rats.Specifically, OLA-induced decrease of leptin and leptin/adiponectin and increase of adiponectin were present in MAM rats only.HAL-induced effect on leptin had the same pattern, showing a decrease after acute dosing.The mixed model did not reveal a significant effect of the main factors (MAM model or treatment) in PAI-1.
The time-dependent analysis showed differential temporal patterns in leptin, FGF-21, and PAI-1 levels.Leptin level was significantly lower 4 h after OLA administration compared to the same timepoint in VEH-treated rats.HAL showed a similar effect 24 h after dosing (trend toward significance, p = 0.057).FGF-21 level was significantly higher 4 h after both OLA and HAL administration compared to the same timepoint in VEH-treated rats.The analysis of PAI-1 data showed a non-specific increase in its levels 4 h after treatment, particularly in the MAM rats.This includes not only the antipsychotics but also VEH treatment, suggesting a non-specific effect exerted by the manipulation rather than pharmacological treatment.

| Serum lipid profile 24 h after drug administration
The complete results of the statistical analysis are presented in Figure 3, together with a graphical representation of the data.In the serum lipid profile, the 2 W ANOVA detected a significant effect of the MAM model in LDL (lower than in CTR rats), TAG, AI, and AIP (higher than in CTR rats).The effect of antipsychotics was found to be significant for all assessed variables.Specifically, OLA decreased the levels of CHOL and HDL and increased AI compared to VEH and/ or HAL treatments.It also increased AIP in comparison with HAL.
HAL increased LDL and decreased TAG (vs VEH treatment).Notably, many of these effects were present in the MAM rats only (results of MAM model*treatment interaction).This applies particularly to the OLA-induced decrease of HDL and increase of AI, which were present only in MAM rats.Similarly, HAL induced an increase in LDL and a decrease in TAG in MAM rats (MAM-HAL vs. CTR-HAL).
Furthermore, OLA increased TAG, AI, and AIP in MAM rats more than in the CTR cohort.

| Two metabolic subgroups among MAM-OLA rats
The data obtained in this study were all found to be normally distributed (except for the ordinal data from the IHC staining).However, when carefully examined, the raw data showed that based on their metabolic response, rats in the MAM-OLA group fell into either of two subgroups: In 4 rats, HDL cholesterol and leptin levels were markedly lower, while TAG and adiponectin levels were higher than in the remaining 5 rats.On the other hand, LDL and total cholesterol values were equal in all rats (Table 1).A sub-analysis distinguishing between responders and non-responders was performed based on these observations.The MAM-OLA rats were deemed responders when the total CHOL minus HDL minus LDL level was equal to or higher than 1 mmol/L (notably, mean values in the CTR-OLA group and non-responders in the MAM-OLA rats were 0.31 mmol/L in both groups).The Kruskal-Wallis test identified multiple significant differences between the MAM-OLA-R (responder) group vs. CTR-OLA and MAM-OLA-NR (non-responder) groups.The same pattern was observed in one MAM-HAL rat but not in any other rats in the experiment.The whole dataset is included in the Supplementary Materials.

| Western blot analysis of the white adipose tissue
A non-parametric approach was used for the statistical analysis of Western blot data -the Kruskal-Wallis test.The only protein showing significant differences among the experimental groups was SCD1: H (5)=16.537, p = 0.006 (Figure 4).A two-tailed test for multiple comparisons revealed a higher amount of the protein in the adipose tissue of MAM-VEH animals compared to the CTR-VEH group (p = 0.042) but no significant effect of antipsychotics.The Kruskal-Wallis test did not indicate significant differences in perilipin (H ( 5

F I G U R E 4
Western blot analysis of SCD1 (37 kDa) in adipose tissue 24 h after treatment.The data are presented as means ±SD and individual data points.The statistical result of the Kruskal-Wallis test is depicted in the graph, *p < 0.05.Note that due to the organization of samples on the gel and the consistency of the graphs in this paper, the experimental groups are organized differently in the graph and the photograph of the gels below it.

| DISCUSS ION
In this study, the acute impact of the metabolically potent antipsychotic OLA and the less metabolically potent drug HAL was compared for a range of metabolic parameters in the MAM neurodevelopmental rat model.The aim of using the MAM model, like other neurodevelopmental models of psychotic disorders, is to produce a phenotype as closely related to first-episode psychosis (FEP) as possible.Indeed, FEP is associated with weight gain and lipid-and glucose-related disturbances present before the initiation of antipsychotic treatment.Altered glucose homeostasis, specifically insulin regulation, and impaired glucose tolerance, as well as derangements in lipid metabolism, are consistently reported in FEP populations, 55 manifesting as hypertriglyceridemia, insulin resistance, and reduced total and LDL cholesterol levels. 56 humans, recent meta-analyses reported increased rates of metabolic syndrome or at least one metabolic syndrome criterion even in drug-naïve FEP, suggesting underestimated cardiovascular risk in this population.With data underlining the notion that altered metabolic parameters are not exclusively associated with antipsychotic exposure, the authors also stressed the need to focus on metabolic risk predictors specific to the FEP population. 57,58Nevertheless, antipsychotic treatment contributes significantly to metabolic derangements in patients, with associated cardiovascular disease risk 1,14 demonstrated even in the absence of psychiatric illness and also preclinically. 59ncerning antipsychotic-naïve metabolic phenotype in schizophrenia-like animal models, only a limited number of studies reported metabolic parameters. 18,36In our previous studies, the MAM model was associated with serum lipid profile alterations in both sexes; total, HDL, and LDL cholesterol levels were increased. 36 another neurodevelopmental model induced by prenatal administration of the polyinosinic:polycytidylic acid (poly I:C), disturbance of lipid metabolism was also observed. 18Our data in MAM and poly I:C models consistently indicate intrinsic lipid alterations even in drugnaïve animals.
Unlike in our previous study, where prenatal MAM exposure did not affect body weight, MAM exposure resulted in lowered body weight in the present cohort, demonstrating the vulnerability induced by MAM exposure. 36However, the alterations in lipid parameters in MAM-treated animals -LDL, TAG, AI, AIP, serum adiponectin, and leptin serum levels and their ratio, as well as protein levels of the fatty acid desaturase Scd1 in white adipose tissue of female rats -are not likely to result solely from reduced body weight.To our knowledge, this is one of the most extensive studies of lipid profile characterization, adipokine, or other peripheral mediator levels in the MAM model. 36us, lipid alterations could represent a trait of neurodevelopmental models reflecting the association of dyslipidemia in antipsychotic naïve FEP patients 60 in accordance with the assumed link between lipid homeostasis and schizophrenia 61 with potential genetic overlap. 62th regard to the effects of antipsychotics, OLA had significantly more pronounced effects in female MAM rats than in non-MAM (CTR) rats for several readouts.Moreover, in the MAM-OLA group, roughly 50% of the animals had a pronounced response to OLA, with serum lipid and adipokine levels significantly deviating from those found in the CTR-OLA group, that is, non-MAM animals treated with OLA.HDL and LDL cholesterol parameters, leptin and leptin/adiponectin ratio were significantly reduced, while TAG, AI, AIP, and adiponectin were massively increased in the MAM-OLA responder group compared to the CTR-OLA group.These effects were in line with previous acute studies in healthy animals, 27,32,42 supporting the current understanding of weight-independent dysregulation of lipid metabolism. 5,63Indeed, preclinical evidence for direct effects of antipsychotics, with consistent findings of increased TAG, possibly reflect a compensatory response to inhibited cholesterol biosynthesis. 5,30,36,63The molecular mechanisms underlying dyslipidemia are still incompletely elucidated; nevertheless, the alterations lead to decreased HDL and increased very low-density lipoprotein and TAG. 5 We carefully considered and eliminated several potential confounders for the subgroup phenomenon (differences in feeding, Data on FGF-21 in schizophrenia are limited.One study reported elevated levels even in first-onset patients with schizophrenia. 65though the FGF system is considered a potential target in schizophrenia, 66 there are no data on the effect of antipsychotics on FGF-21. 67FGF-21, as a mediator of energy homeostasis, represents a target for emerging new potentially beneficial therapies for obesity, dyslipidemia, and glucose dysregulation [68][69][70] and possibly in the development of strategies in prevention or treatment of adverse metabolic effects of antipsychotics.
The present study was conducted in female rats.Recently reviewed clinical and preclinical data regarding sex differences in antipsychotic-associated metabolic disturbances report females at higher risk of weight gain and diagnosis of metabolic syndrome.
However, accumulation of adipose tissue depots and glucose homeostasis dysregulation also occur in male rats and are considered independent of weight changes. 27Resonating with this, while male rats do develop features such as adiposity, lipogenic activation, and adverse lipid profile in the absence of weight gain, female rats are more susceptible to antipsychotic-induced weight gain and related dysmetabolic features. 8,71In our previous experiments, sexdependent alterations in lipid profile were found; the female sex was associated with higher total cholesterol and HDL, whereas LDL, AI, AIP, and CRP were higher in male rats in both control and MAM cohorts. 36In summary, female rats are considered more relevant for modeling antipsychotic-associated weight gain than male rats, concerning both face and construct value.Therefore, the female model was utilized in the present experiment.

| CON CLUS IONS
In summary, our results demonstrate that prenatal MAM exposure led to a metabolic vulnerability in female rats, with pronounced effects of OLA in approximately 50% of the MAM cohort and less pronounced HAL effects in accordance with the respective drugs' degree of clinical impact on metabolism. 1,4However, a central issue affecting the validity of the MAM model is whether MAM treatment yields metabolic effects comparable to those found in schizophrenia/psychosis. Determining a clear pattern of the metabolic profile of these psychiatric disorders is complicated, as numerous studies did not take antipsychotic medication unequivocally into account, as many patients in the studies were not treatment naïve.

Statistics 28 .
0.0.0(190).A value of p < 0.05 was set as the boundary of statistical significance in all applied tests.The data were tested for normality by the Shapiro-Wilk test and analyzed by a parametric or non-parametric method as appropriate.The study design features two factors -the MAM model (CTR-MAM) and treatment (VEH-OLA-HAL).For body weight and serum lipid analysis, a two-way (2 W) ANOVA was performed with Tukey's post-hoc test when significant differences were detected in the treatment factor or model*treatment interaction.To analyze a repeated sampling of serum adipokine levels (ELISA), a repeated measures approach was selected to test the effect of the MAM model and treatment in three consecutive measurements.The mixed-effects model was used with Sidak's post-hoc test when appropriate.For Western blot analysis of adipose tissue and immunohistochemical staining of liver slices, a non-parametric approach was used -the Kruskal-Wallis test.For the subgroup analysis of selected biochemical variables (CHOL, HDL, LDL, TAG, AI, AIP, leptin, adiponectin, and L/A ratio) in CTR-OLA and MAM-OLA rats, where responders and non-responders were found in the MAM-OLA group, the Kruskal-Wallis test was used due to a low number of subjects (n = 4-5).
The liver histological examination was performed to determine pathological changes induced by prenatal MAM exposure.No difference in inflammatory signs, steatosis, or fibrosis was detected as an index F I G U R E 2 Adipokine levels at 1, 4, and 24 h after treatment.The data are presented as means ±SD and individual data points.The tables show the results of the mixed-effects model and Sidak's post-hoc test where applicable.F I G U R E 3 Serum lipids at 24 h after treatment.The data are presented as means ±SD and individual data points.The tables show the 2 W ANOVA and Tukey's post-hoc test results where applicable. of pathological changes between the MAM and CTR groups.Given the entirely physiological state of the tissue, the photographs are not presented.For the immunohistochemical staining, the Kruskal-Wallis analysis revealed a significant FGF-21 immunopositivity in the liver tissue: H (5)=15.144, p = 0.010.A two-tailed test for multiple comparisons identified a significantly higher FGF-21 immunopositivity in MAM-VEH animals compared to MAM-HAL (p = 0.039).The data are shown in Figure 5.No differences were detected in the levels of PAI-1 in the liver tissue as the test showed a significant result (H (5)=12.696,p = 0.026).However, the test for multiple comparisons did not reveal any significantly different experimental groups.Immunohistochemical staining of IL-1beta and IL-6 in the liver tissue revealed too low, barely detectable expression levels in all groups.TA B L E 1 OLA-induced metabolic response in MAM rats.
Nevertheless, the noticeable impact of a neurodevelopmental model on baseline lipid metabolism and the metabolic response to OLA underlines that findings from healthy rats are likely to provide an underestimated impression of metabolic dysfunction.Neurodevelopmental models possess the most relevant background in the context of metabolic disturbances.The search for adequate animal models should receive more attention within the field of experimental psychopharmacology.AUTH O R CO NTR I B UTI O N S KH prepared the design and protocol of the study and contributed to writing the manuscript.SS initiated the interpretation of the results and contributed to writing the manuscript.JK processed the samples and conducted the laboratory analyses (Western blot).GK processed the samples and conducted laboratory analyses (histology and immunohistochemistry).PS coordinated the laboratory analyses.VM developed the MAM model and prepared the animals for the study.JRK planned the experiments, conducted the in vivo part of the study, collected all data, performed the statistical analysis, prepared figures and tables, and contributed to writing the manuscript.All authors revised and edited the final manuscript.